A colostrum casein TG enzyme-induced complex capable of reducing postprandial glycemic index and a preparation method thereof

Abstract

This invention discloses a bovine colostrum casein TG enzyme-induced complex and its preparation method, which can reduce postprandial glycemic index, belonging to the field of functional food and biomolecular modification technology. Specifically, the bovine colostrum casein TG enzyme-induced complex is prepared by dissolving bovine colostrum casein in water to form a casein solution, then adding a complexing component and stirring to dissolve; subsequently, TG enzyme is added to carry out a cross-linking reaction, and the solution is dried after the reaction. This complex is a supramolecular aggregate with a "cavitary structure," "multiple binding sites," and "physical retention capacity." After entering the gastrointestinal tract, it efficiently adsorbs / complexes monosaccharides, disaccharides, and starch hydrolysis intermediates in the diet through various non-covalent interactions such as hydrogen bonding, hydrophobic interactions, and ion complexation, physically delaying their diffusion and absorption into the small intestinal epithelium, thereby reducing postprandial blood glucose peaks and fluctuations, and possessing the potential to be developed into an adjunctive hypoglycemic functional preparation.

Description

Technical Field

[0001] This invention relates to a bovine colostrum casein TG enzyme-induced complex that can reduce postprandial glycemic index and its preparation method, belonging to the field of functional food and biomolecular modification technology. Background Technology

[0002] Postprandial hyperglycemia is a typical metabolic abnormality in patients with prediabetes and type 2 diabetes, and is also an independent risk factor for cardiovascular complications. Current strategies for delaying postprandial blood glucose elevation mainly include: using alpha-glucosidase inhibitors (such as acarbose) to delay carbohydrate digestion, or increasing chyme viscosity through dietary fiber to physically block sugar absorption.

[0003] However, existing technologies have significant shortcomings: α-glucosidase inhibitors easily cause gastrointestinal side effects such as bloating and diarrhea; dietary fiber products generally have weak adsorption capacity for sugar molecules and poor selectivity. Therefore, developing a functional ingredient that can both efficiently "capture" sugar molecules and is safe and edible has important application value.

[0004] Bovine colostrum casein is rich in various functional domains and active groups, possessing a natural structural advantage as a carrier for sugar molecule adsorption, making it a potential functional matrix for blood glucose regulation. However, natural casein has a low affinity for sugar molecules and limited adsorption capacity, failing to achieve significant postprandial blood glucose inhibition effects and thus failing to meet practical application requirements. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a bovine colostrum casein TG enzyme-induced complex that can reduce postprandial glycemic index and its preparation method. The preparation of this complex utilizes TG enzyme catalysis to cross-link / polymerize bovine colostrum casein with specific complexing components, forming a macromolecular complex with a three-dimensional network structure. This complex is a supramolecular aggregate with a "cavitary structure," "multiple binding sites," and "physical retention capacity." After entering the gastrointestinal tract, through various non-covalent interactions such as hydrogen bonding, hydrophobic interactions, and ion complexation, it efficiently adsorbs / complexes monosaccharides, disaccharides, and starch hydrolysis intermediates in the diet, physically delaying their diffusion and absorption into the small intestinal epithelium, thereby reducing postprandial blood glucose peaks and fluctuations. It has the potential to be developed into an adjunctive hypoglycemic functional preparation.

[0006] To achieve the above objectives, the following technical solution is provided: This invention provides a method for preparing a bovine colostrum casein TG enzyme-induced complex that can reduce the postprandial glycemic index, comprising the following: Bovine colostrum casein is dissolved in water to form a casein solution. Then, the complexing component is added and stirred to dissolve. After that, TG enzyme is added to carry out a cross-linking reaction. After the reaction, the solution is dried to obtain the final product.

[0007] In one embodiment, the bovine colostrum casein refers to: collecting bovine colostrum within 72 hours after calving, defatting it, and separating crude casein by acid precipitation (pH 4.6) or rennet method; reconstituted the crude product and purified it by ultrafiltration, spray drying or freeze drying to obtain bovine colostrum casein powder with a purity ≥90%.

[0008] In one embodiment, the casein solution has a mass fraction of 6%-12%.

[0009] In one embodiment, the complex component includes one or more of chitosan (molecular weight 3 kDa-50 kDa), carboxymethyl chitosan, sodium alginate, low-ester pectin, and chondroitin sulfate.

[0010] In one embodiment, the mass ratio of the complex component to casein is 1:5 to 2:1; the amount of complex component added affects the network density and adsorption capacity of the complex; if the ratio is too low (<1:5), the gel network will be imperfect; if the ratio is too high (>2:1), the system will be too viscous and difficult to disperse, and may reduce the selective adsorption of neutral sugars.

[0011] In one embodiment, the TG enzyme is added at an amount of 1-15 U / g casein.

[0012] In one embodiment, the pH of the crosslinking reaction system is 5.5-7.0; the temperature is 30-50℃; the reaction time is 30-180 minutes; and the stirring speed is 100-300 rpm. A pH of 5.5-7.0 is beneficial for maintaining high activity of TG enzymes, while avoiding precipitation of casein near its isoelectric point, and also facilitates the dissolution of complex components (such as chitosan). The reaction time affects the degree of crosslinking and the molecular weight distribution of the complex. The endpoint can be determined by monitoring the upward shift of the casein band using SDS-PAGE: when the native casein band (approximately 25-35 kDa) essentially disappears and residue appears at the top of the lane, it indicates sufficient crosslinking; if the crosslinking is too low, the complex will have insufficient adsorption capacity for sugars; if the crosslinking is too high, it may form insoluble large gel masses, affecting dispersibility in the gastrointestinal tract.

[0013] In one embodiment, the reaction is followed by heating to 75-85°C and holding for 10-20 minutes to inactivate the TG enzyme.

[0014] In one embodiment, the drying is carried out by spray drying (inlet air temperature 160-180℃, outlet air temperature 70-85℃) or freeze drying to obtain a powdered product; spray drying can maintain the good solubility and dispersibility of the product, which is conducive to its rapid hydration in gastrointestinal fluid to form an active adsorbed form.

[0015] The present invention also provides a bovine colostrum casein TG enzyme-induced complex prepared by the method described above.

[0016] The present invention also provides the application of the bovine colostrum casein TG enzyme-inducible complex described above in the preparation of products for reducing postprandial glycemic index.

[0017] In one embodiment, the product includes functional foods, health foods, special medical foods, or pharmaceuticals.

[0018] The present invention also provides a food composition for reducing postprandial glycemic index, comprising the bovine colostrum casein TG enzyme-inducible complex described above and a food-grade acceptable carrier.

[0019] In one embodiment, the food composition is taken before or with a meal, with a single dose of 2-15 g based on casein.

[0020] Technical principle of the invention: Unlike traditional covalent binding or enzymatic inhibition mechanisms, the mechanism by which the complex constructed in this invention lowers postprandial blood glucose is as follows: (1) Spatial network trapping effect: TG enzyme induces cross-linking between bovine colostrum casein and complex components (such as chitosan, sodium alginate or pectin) to form macromolecular complexes with a three-dimensional loose network structure. These complexes remain in particulate or colloidal state in the gastric acid environment and can physically encapsulate or trap starch, dextrin and oligosaccharides ingested at the same time; (2) Multivalent adsorption / complexation: The surface of the polymer is rich in hydrophobic regions of casein and phosphoserine clusters (Ca²⁺). + The binding sites and functional groups such as hydroxyl, carboxyl, and amino groups in the complex components. These groups can bind to the hydroxyl groups of sugar molecules (especially glucose and maltose) at multiple sites through non-covalent bonding forms such as hydrogen bonding, hydrophobic interactions, cation-π interactions, and ion complexation. Because it is a non-covalent bond, the sugar molecules maintain a dynamic equilibrium of "bound state - free state" in the intestine, thereby continuously and gradually releasing free sugars and avoiding a large amount of sugar entering the bloodstream at once.

[0021] (3) Delaying gastric emptying and increasing the thickness of the unstirred layer: The weak gel state formed by the polymer in the stomach can moderately delay the gastric emptying rate; after entering the intestine, the unstirred layer adsorbed on the surface of the intestinal mucosa increases the resistance to the diffusion of sugar molecules to the brush border.

[0022] The complex described in this invention does not rely on chemical modification of the sugar molecules themselves, nor does it inhibit the enzyme activity of α-glucosidase. Instead, it delays the release and absorption of sugar through physical isolation and adsorption complexation mechanisms. This mechanism determines that it has a mild effect, no bloating side effect common to enzyme inhibitors, and has a delaying effect on the absorption of various types of sugars (monosaccharides, disaccharides, oligosaccharides).

[0023] In this invention, TG enzyme is used as a "structure building tool". TG enzyme catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide bonds between glutamine residues and lysine residues in bovine colostrum casein molecules, enabling casein molecules to form larger oligomers or network backbones. At the same time, TG enzyme can graft polysaccharides such as chitosan containing primary amine groups in the complex component onto the casein backbone, enhancing the functional group density and charge properties of the complex. The resulting complex has a significantly increased physical affinity for sugar molecules due to the significant increase in molecular weight, exposure of hydrophobic regions, and changes in charge properties.

[0024] Beneficial effects: (1) This invention innovatively utilizes TG enzyme to construct a three-dimensional network of bovine colostrum casein-complex components, forming a highly efficient physical adsorption / complexation system for sugar molecules. The mechanism is different from that of enzyme inhibitor hypoglycemic products, thus avoiding the side effect of bloating. (2) By selecting bovine colostrum casein and chitosan and other complex polymer components, the present invention obtains a functional complex polymer with high sugar adsorption capacity and excellent dispersibility in gastrointestinal fluid under cross-linking conditions precisely controlled by TG enzyme. (3) In vitro experiments show that the adsorption rate of glucose by the polymer of the present invention is more than 4 times higher than that of physical mixtures; animal experiments show that it can reduce the peak blood glucose level after meals by about 35%, which is comparable to the effect of acarbose but has better safety.

[0025] (4) The product of this invention is in powder form and can be added to food carriers such as beverages, dairy products, and meal replacement powders, with a wide range of applications. Attached Figure Description

[0026] Figure 1 The image shows the physical composite prepared in Comparative Example 3. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0028] The source of raw materials involved in this invention: Bovine colostrum casein: The raw material was purchased from Heilongjiang Kangping Biotechnology Co., Ltd.; Regular casein: The raw material was purchased from Heilongjiang Kangping Biotechnology Co., Ltd.; Low-ester pectin: Raw materials were purchased from Sichuan Huanxu Biotechnology Co., Ltd.

[0029] Example 1 A method for preparing a bovine colostrum casein TG enzyme-induced complex that lowers the postprandial glycemic index includes the following: (1) Take 10 g of bovine colostrum casein, dissolve it in 100 mL of deionized water, adjust the pH to 6.0, and stir for 2 hours until completely dispersed; (2) Add 2g of chitosan (85% deacetylation, 10 kDa molecular weight) to the solution dispersed in step (1), stir to dissolve, then add TG enzyme (100 U / g activity) at a rate of 5 U / g casein, stir at 40℃ for 90 min, inactivate the enzyme in a water bath at 80℃ for 15 min, cool, freeze dry to obtain the polymer.

[0030] In vitro sugar adsorption experiment: 1g of the polymer powder was added to 50mL of 5% glucose solution (simulating postprandial glucose concentration) and maltose solution, respectively, and shaken at 37℃ for 30 minutes; after centrifugation, the supernatant was collected, and the residual glucose concentration was determined by glucose oxidase method; an untreated bovine colostrum casein + chitosan direct physical mixture was used as a negative control, and an equal amount of activated carbon was used as a positive control. The results are shown in Table 1: Table 1. Adsorption rate

[0031] The results show that the adsorption rate of glucose by the polymer of this invention is nearly 4 times higher than that of the physical mixture, indicating that the TG enzyme-mediated crosslinking significantly enhances the system's ability to bind sugar molecules.

[0032] Example 2 Following the method of Example 1, the chitosan in step (2) was replaced with low-ester pectin, sodium alginate, and carboxymethyl chitosan, respectively, in the following specific ratios (mass ratios): Example 2A: Low-ester pectin (bovine colostrum casein: pectin = 2:1) Example 2B: Sodium alginate (bovine colostrum casein:sodium alginate = 3:1) Example 2C: Carboxymethyl chitosan (bovine colostrum casein:carboxymethyl chitosan = 4:1); other parameters and conditions are the same as in Example 1.

[0033] The results of the in vitro sugar adsorption experiment are shown in Table 2; Table 2. Adsorption rate

[0034] Comparing the data from Example 1 and Example 2, it was found that the complex containing aminopolysaccharides (chitosan, carboxymethyl chitosan) has a stronger adsorption capacity for sugars, which may be related to the electrostatic interaction and the higher grafting efficiency mediated by TG enzyme.

[0035] Comparative Example 1 The difference from Example 1 is that TG enzyme is not added; bovine colostrum casein is simply physically mixed with chitosan and then dried.

[0036] As shown in Table 1 above, the glucose adsorption rate was only 12.3%, significantly lower than that in Example 1. This demonstrates that the TG enzyme-induced cross-linking structure is key to improving adsorption capacity.

[0037] Comparative Example 2 The difference from Example 1 is that commercially available regular milk casein (derived from regular milk) is used instead of bovine colostrum casein, while other parameters and conditions remain unchanged.

[0038] The results of the in vitro sugar adsorption experiment are shown in Table 3. Table 3. Adsorption rate

[0039] The above results indicate that, under identical TG enzyme cross-linking processes and with the same complexing component (chitosan), the 30-minute adsorption rates of glucose and maltose by the casein complex derived from bovine colostrum reached 58.7% and 43.6%, respectively, significantly higher than the 31.4% and 19.6% of ordinary bovine colostrum casein complex. The former's adsorption capacity for the two sugars was increased by approximately 86.94% and 122.45%, respectively. This difference is not accidental but is determined by the fundamental differences in molecular structure and functional groups between bovine colostrum casein and ordinary casein; the reasons are as follows: First, bovine colostrum casein exhibits a higher degree of phosphorylation. The density of phosphoserine (Ser-P) clusters in bovine colostrum casein micelles is significantly higher than that in regular milk. Phosphoserine residues not only form additional ionic bridges through calcium ions, enhancing the three-dimensional network stability of the polymer, but more importantly, the phosphate groups can act as multivalent donors / acceptors of hydrogen bonds, forming multi-site hydrogen bond networks with the hydroxyl groups of sugar molecules (especially glucose). In this study, the increase in maltose adsorption rate of the bovine colostrum polymer (122.45% higher) was even greater than that of glucose (86.94%), suggesting that the synergistic effect of multivalent hydrogen bonds is more significant for disaccharides containing more hydroxyl groups.

[0040] Second, bovine colostrum casein is rich in naturally occurring bioactive peptides and hydrophobic regions. During enzymatic hydrolysis or processing, bovine colostrum casein more readily exposes hydrophobic amino acid residues (such as leucine, isoleucine, and proline). These hydrophobic regions can form nonpolar bonds with the secondary hydroxyl groups (axial and equatorial positions) of sugar molecules through hydrophobic interactions. In contrast, ordinary casein, due to its more compact structure, has insufficient exposure of hydrophobic regions. After cross-linking with TG enzymes, the surface of the complex formed by bovine colostrum casein exhibits a more pronounced hydrophobic / hydrophilic amphiphilic interface. This interface structure facilitates the simultaneous adsorption of sugar molecules of different polarities, thereby expanding its broad-spectrum binding capacity for both monosaccharides and disaccharides.

[0041] Third, bovine colostrum casein contains a higher proportion of glutamine (Gln) and lysine (Lys) residues, which are the substrates for TG enzymes. Under the same TG enzyme addition (5 U / g protein) and reaction conditions, bovine colostrum casein exhibits a significantly higher degree of cross-linking (which can be determined by the proportion of protein retained in the sample wells on SDS-PAGE: approximately 86% in the bovine colostrum group and approximately 52% in the regular milk group). This higher degree of cross-linking means the formation of a denser yet porous three-dimensional network structure, which provides a larger specific surface area to expose binding sites and also offers appropriately sized "molecular cages" to physically trap larger sugar molecules such as maltose.

[0042] In summary, the unique phosphorylation level, abundance of active groups, and TG enzyme activity of bovine colostrum casein collectively endow the final polymer with a sugar adsorption capacity far superior to that of ordinary casein.

[0043] Comparative Example 3 The difference from Example 1 is that the amount of TG enzyme added is increased to 30 U / g casein, the reaction time is extended to 240 minutes, and other conditions and parameters are the same as in Example 1.

[0044] The results showed that the reaction system formed insoluble large gels (such as...). Figure 1 As shown in the figure, after drying, the powder has extremely poor dispersibility (<20%) and poor suspension in simulated gastric fluid, resulting in a significant decrease in actual adsorption effect.

[0045] Example 3: Verification of hypoglycemic effect in animals (oral maltose loading test in rats) Animals: Male SD rats, 8 weeks old, n=48, randomly divided into 4 groups (n=12 per group). Group A: Blank control (maltose loading + physiological saline) Group B: Acarbose positive control (25 mg / kg) Group C: Complex from Example 1 (500 mg / kg, based on casein) Group D: Bovine colostrum casein + chitosan physical mixture (500 mg / kg); Modeling: Fasting for 12 hours before the experiment, administer the test substance or control solution by gavage first, and then administer a maltose loading (2 g / kg) 15 minutes later. Tests: Blood was collected from the tail vein and blood glucose was measured at 0, 15, 30, 45, 60, 90, and 120 minutes. The results are shown in Figure 4. Table 4. Blood Glucose Changes

[0046] Blood glucose levels obtained from tail vein sampling showed no significant difference in initial fasting blood glucose levels among the groups, with consistent baseline levels, thus eliminating the interference of initial blood glucose differences on the experimental results. After modeling treatment, the postprandial blood glucose in the blank group peaked at 30 min and increased significantly, maintaining high blood glucose levels at 60 min and 120 min, with large fluctuations in blood glucose. Both the acarbose positive control group and the complex polymer group of this invention significantly inhibited the rapid rise in postprandial blood glucose and effectively reduced the peak blood glucose level. The complex polymer of this invention reduced the peak blood glucose level at 30 min by 33.6%, a hypoglycemic effect similar to the 36.7% peak reduction in the acarbose positive control group, demonstrating excellent postprandial blood glucose regulation ability. The physical mixing group only achieved a 12.5% ​​peak reduction, with a weak hypoglycemic effect, significantly worse than the complex polymer of this invention, indicating that the special complex structure of this invention is not a simple physical superposition, but rather exerts hypoglycemic activity through synergistic effects between components. Meanwhile, no gastrointestinal adverse reactions such as diarrhea or bloating occurred in the test animals throughout the entire experimental period, indicating that the polymer of the present invention has good safety and has the potential to be developed into a functional preparation for adjuvant hypoglycemia.

[0047] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing a bovine colostrum casein TG enzyme-inducible complex that can lower the postprandial glycemic index, characterized in that, The method includes the following: Bovine colostrum casein is dissolved in water to form a casein solution, then a complexing component is added and stirred to dissolve; then TG enzyme is added to carry out a cross-linking reaction, and the solution is dried after the reaction to obtain the final product; the complexing component includes one or more of chitosan, carboxymethyl chitosan, sodium alginate, low-ester pectin, and chondroitin sulfate.

2. The method according to claim 1, characterized in that, The casein solution has a mass fraction of 6%-12%.

3. The method according to claim 1, characterized in that, The mass ratio of the complex component to casein is 1:5 to 2:

1.

4. The method according to claim 1, characterized in that, The TG enzyme is added at a rate of 1-15 U / g casein.

5. The method according to claim 1, characterized in that, The cross-linking reaction system has a pH of 5.5-7.0, a temperature of 30-50℃, a reaction time of 30-180 minutes, and a stirring speed of 100-300 rpm.

6. The method according to claim 1, characterized in that, The drying process employs spray drying or freeze drying to obtain a powdered product.

7. The bovine colostrum casein TG enzyme-induced complex prepared by the method according to any one of claims 1 to 6.

8. The use of the bovine colostrum casein TG enzyme-inducible complex of claim 7 in the preparation of a product for reducing postprandial glycemic index.

9. The application according to claim 8, characterized in that, The products include functional foods, health foods, special medical foods, or medicines.

10. A food composition for lowering the postprandial glycemic index, characterized in that, It includes the bovine colostrum casein TG enzyme-inducible complex of claim 7 and a food-grade acceptable carrier.

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